Method and apparatus for biomolecule analysis

ABSTRACT

Disclosed are devices or apparatus and methods for membrane-, bead- or cell-based biomolecule analysis, where a detectable signal can be measured after affinity binding and/or enzymatic reaction occurs on the membranes, beads or cells. The apparatus or device can rapidly separate the beads or cells from the liquid solution without using centrifugation, vacuum, or magnetic force. The apparatus or device includes a sample well for receiving a sample containing cells or beads, as well as other aqueous solutions, a porous filter membrane at the bottom of the well which is capable of retaining the cells or beads on its upper surface by size exclusion, and an absorbing plug in touch with the filter membrane for removing the liquid solution. Multiple devices can be arranged in a multi-well array in a plate so that signals from multiple assays can be directly analyzed in a plate reader or an imaging instrument.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/662,117, filed on Mar. 18, 2015, which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Application No. 61/994,645, filed onMay 16, 2014, which is incorporated by reference in its entirety.

FIELD

Disclosed are novel apparatus and uses thereof in biomolecule analysis.Particular embodiments contemplate membrane, cell or bead-basedbiomolecule analysis, where after a biochemical reaction such as anenzymatic reaction or an affinity binding, the signals carried by themembranes, cells or beads are to be analyzed to determine the result ofthe assay.

BACKGROUND

Microspheres, microparticles, or beads, have become an important form ofsolid state substrate for detecting, analyzing and identifying a widerange of biomolecules. Advantages of using microspheres compared totraditional plate-based assay methods include their large surface area,easiness of mixing with a solution and re-collecting, availability ofdifferent chemistry to functionalize the beads and control the densityand orientation of the reactive ligands on the beads. Preparation of thebeads can be easily scaled up with consistent quality of beads.Different types of microspheres can be selected based on specificrequirements of the assay protocol and signal detection method. Amongthe most widely used microspheres, there are silica beads, magneticbeads, polystyrene beads and latex beads.

In a typical bead-based assay, for example, magnetic bead-basedimmuno-assay, magnetic microspheres functionalized with a specificcapture antibody will be first incubated with a fluid sample in whichthe suspect antigen may present, and then a solution of detectionantibody conjugated with an enzyme, and finally solution of enzymesubstrate to generate a detectable signal. During the process, inbetween different incubations, stringent washing of the beads arecritical for accurate results. Bead washing is normally accomplished byrepeated cycles of bead collection, aspiration, and re-suspension,through centrifugation, magnetic aggregation, or vacuum assistedfiltration. All of these methods are lengthy, repetitive, and need extraequipment operation. After the assay, recollecting and measuring thesignals of the microspheres is also a big challenge, especially forassays using small amount of beads and multiple washing steps, becauseloss of beads is inevitable in most cases. Similar protocols are usedfor many cell-based assays to examine the biomolecules on the cellmembrane or inside the cells. Commonly used lab tools include multiwellfiltration devices which utilize vacuum or centrifugation force toextract the liquid out of the filtration well.

Dot blot is another widely used membrane-based biomolecule analysismethod. In this format, typically, one or more specific biomoleculessuch as antibodies, antigens, and nucleic acids, are immobilized on aporous membrane in an array of small confined area. The blotted membraneis then placed in a serial of buffer and solutions for membraneblocking, affinity binding, rinsing, and signal development. The entireprocess, including multiple lengthy shaking and washing steps, takesmore than two hours or even overnight to finish. A commonly usedapparatus for dot blot assay relies on a vacuum source to suck solutionthrough the membrane, but the apparatus set up is complicated,expensive, and need to be assembled, disassembled and cleaned for everyuse. Only a limited number of membranes can be processed using suchdevice. Cross-contamination is hard to avoid in these methods becauseone continuous piece of membrane is used for multiple assay dots.Therefore, there are needs in the art for apparatus and methods that cansimplify the washing and shorten the total assay time for both highthroughput analysis platform and rapid test for a few samples inresearch and clinical labs.

SUMMARY OF THE INVENTION

The present disclosure describes an apparatus or a device and its usagefor quick and efficient collection of beads and wash of the beads andfor signal detection in a bead-based biomolecule analysis. The sameapparatus or device can also be applied for cell-based analysis andmembrane-based dot blot assays. Advantageously, the apparatus andmethods of the present disclosure simplify the washing step withoutusing centrifuge, vacuum, or magnetic force; provide more efficientwashing with less volume of buffer and less time; are capable ofcondensing the beads and biomolecules in a small detection area togenerate stronger signal; use significantly less amount of assay reagentthan traditional assays; and enable quantitative signal detection andanalysis using a plate reader or an imaging device.

In certain embodiments, each apparatus comprises one or more individualdevices assembled in a plate. Each device has a sample well with anupper opening, bottom opening, and an inner wall, such as a sloped wallor a tapered wall for a user to introduce the bead suspension, samplesolutions and additional solution into the device, and for the beads toaggregate in the detection zone at the bottom of the well for detection.

A filter membrane covers the bottom opening of the sample well.Microspheres can be retained on the upper surface of the filter membraneby size-exclusion. In some embodiments, biomolecules can be immobilizedon the membrane by adsorption or other means. Preferably, the filtermembrane is made of porous hydrophilic material so that aqueoussolutions containing biomolecules can pass through the membrane bycapillary force and gravity, without other external force.

The device further contains an absorbing plug placed in close contactwith the filter membrane. The absorbing plug is made of hydrophilicporous material, which allows its rapid absorbance of aqueous solutionthrough the filter membrane into the plug by capillary force.

Each device is individually enclosed and therefore cross-contaminationis totally eliminated. Multiple devices can be arranged in an arrayformat, such as the ANSI/SLAS 2-2004 standard 96 well format. For moreflexible use, one or multiple devices pre-assembled together in an arraystrip or block can be inserted into a plate frame by the user.

A bead-based or cell-based assay can first be performed in a separatecontainer for necessary mixing, incubation and any other necessaryprocess. The user can then transfer the solution containing the beads orcells to the device. Because of the tapered wall of the sample well, thebeads will aggregate at the bottom of the well while the solution isabsorbed by the filter membrane and the absorbing plug by capillaryforce. To rinse off the unbound molecules from the beads or cells, acertain volume of the rinsing buffer is added to the well, and it willbe absorbed by the absorbing plug. Multiple washing steps can beperformed as long as the total volume of solution added into the devicedoes not exceed the maximum pore volume or absorbing capacity of theabsorbing plug. Besides washing, additional solution such as enzymesubstrates can be added into the sample well to allow reaction to occuron the beads or cells inside the detection zone. Optionally, to reducenon-specific binding, the user can add blocking buffer onto the filtermembrane before introducing the beads or cells into the device.

In a membrane-based assay, the user can first blot the agent to beimmobilized onto the membrane inside the sample well and let it dry.Afterwards, the user can sequentially add blocking buffer and otherreaction solutions such as antibodies and enzyme substrates into thewell and let them go through the blotted membrane by capillary force andreact with the immobilized molecules. Incubation time can be adjusted bythe user for optimal result.

In cell, bead, or membrane-based assays using the device as described inthis invention, multiple samples and plates can be processed at the sametime manually or automatically. At the end of the assay, the entireplate can be analyzed visually or by a signal detecting means orinstrument such as plate reader or imaging instrument. The used devicesare not reusable and can be disposed properly.

The features, aspects, and advantages of the present invention willbecome better understood with reference to the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure, as defined in the claims, can be betterunderstood with reference to the following drawings. The drawings arenot all necessarily drawn to scale, emphasis instead being placed uponclearly illustrating principles of the present invention.

FIG. 1 is a schematic illustration of the cross-section of a singledevice unit.

FIGS. 2A-2E are schematic illustrations of examples of differentgeometric designs of the absorbing plugs in a single device.

FIGS. 3A-3F are schematic illustrations of examples of arrangement ofmultiple devices in a plate format. FIG. 3A: 8×12 array, 96 well wholeplate. FIG. 3B: 8×12 array, 96 well plate individual insert. FIG. 3C:8×1 strips in 96 well plate frame. FIG. 3D: 1×12 strips in 96 well plateframe. FIG. 3E: 6×8 array, 48 well plate format, FIG. 3F: 4×6 array, 24well plate format.

FIG. 4 is a schematic illustration of how the signal generated in theapparatus after a biological assay can be detected by a plate reader.

FIG. 5 is an embodiment of the invention.

FIG. 6 is a schematic illustration of the process of a bead-basedfluorescent immunoassay performed using the apparatus as described inthe present disclosure.

FIG. 7 is a schematic illustration of the process of a bead-basedchemiluminescent immunoassay performed using the apparatus as describedin the present disclosure.

FIG. 8 is a schematic illustration of the process of a bead-basedfluorescent BoNT/A activity detection assay using the apparatus asdescribed in the present disclosure.

FIG. 9 is a calibration curve obtained with a bead-based assay using theapparatus as described in the present disclosure.

FIGS. 10A and 10B are schematic illustrations of the process of achemiluminescent dot blot assay using the apparatus as described in thepresent disclosure. FIG. 10A: the spot, block and probe steps in thechemiluminescent dot blot assay. FIG. 10B: the incubation, rinsing anddetect steps in the chemiluminescent dot blot assay.

FIG. 11 is a chemiluminescent image resulting from a dot blot assayusing the apparatus as described in the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is related to laboratory apparatus used inbiomolecule analysis on cell, beads, or porous membranes. After abiochemical reaction such as an enzymatic reaction or an affinitybinding, the signals carried by the membranes, cells or beads are to beanalyzed to determine the result of the assay.

As shown in FIG. 1, an individual device 78 described in this inventioncomprises a sample well 30, a filter membrane 10, and an absorbing plug20. All three parts are assembled together inside a housing 14.

The sample well 30 is for addition of sample and other solutions intothe device. The dimension of the well will vary for differentapplications but typically ranges from 3 to 30 mm in diameter and 3 to30 mm in height. In some embodiments, the dimension of the well is from6 mm to 18 mm in diameter and 4 mm to 10 mm in height. The cross-sectionof the well can be circular, elliptical, square, rectangular,pentagonal, hexagonal, octagonal, or any other shapes. The well has asloped inner wall 31 to guide the solution flow toward the bottom of thewell by gravity. The bottom of the well is open to the second componentfilter membrane 10, and forms a detection zone 12 for the beads toaggregate, or in some embodiments, for the biomolecules to beimmobilized on the filter membrane. The detection zone 12 is preferablycylindrical shaped to ensure a uniformed distribution of the beads whenthey aggregate on top of the filter membrane 10. In preferredembodiments, the detection zone is 0.5-7 mm in diameter and 0.5-2 mm inheight. The diameter and height of the detection zone can be varied tohold different volume of beads for different applications. The well ismade of a low fouling material such as a polymer or a copolymer, e.g.,polypropylene or poly(methyl methacrylate) (PMMA) to minimizenon-specific biomolecule adhesion. The well can be transparent oropaque. For fluorescence and luminescence detection, opaque material ispreferred to minimize external light interference.

The second component 10 is a filter membrane, or a porous membrane,whose pore size is selected to be smaller than the size of themicrospheres to be used in a bead-based bioassay. The filter membrane iscapable of preventing the passage of microspheres whose diameters arebigger than the filter membrane's pore size, and at the same timeallowing the passage of the aqueous solution and particles smaller thanthe filter membrane's pore size. In some embodiments, the membrane has apore size between about 0.3 μm to about 50 μm. The pores in the membranecan be randomly or uniformly distributed. In one embodiment, themembrane has pores with a uniform size with a certain variance. Anyhydrophilic porous organic or inorganic material can be used as thefilter membrane, such as silver membrane, glassfiber membrane,nitrocellulose membrane, mixed cellulose ester (ICE) membrane,polycarbonate (PC) membrane, polyester (PET) membrane, cellulose acetatemembrane, nylon membrane, polyethersulfone (PES) membrane,polyvinylidene difluoride (PVDF) membrane, regenerated cellulose (RC)membranes etc. Some intrinsically hydrophobic material can be treated tobecome hydrophilic, such as porous polyethylene (PE) and polypropylene(PP), and can also be used as filter membrane. Thickness of the membranecan be from about 10 μm to 10 mm. In some embodiments, the thickness ofthe membrane is from about 100 to about 1 mm. The terms “pore” and “voidspace” are used interchangeably.

The filter membrane can be used without further treatment, or can befurther processed to alter its surface property. In certain embodiments,to facilitate the passage of free bio-molecules such as proteins andpeptides through the filter membrane, the filter membranes have lowprotein binding surface. Membranes made of low fouling materials, suchas polyethersulfone (PES), cellulose acetate, and regenerated cellulose(RC), can be used directly without additional treatment. In certaininstances, such membranes and other membranes with higherprotein-binding capabilities, such as glassfiber, nitrocellulose, nylon,and mixed cellulose ester (MCE), can be treated with bovine serumalbumin (BSA) or other commercially available blocking solutions toreduce non-specific protein adsorption.

In one instance, the disc filters are first soaked with Superblockblocking buffer from Thermo Scientific for 8 hours and then rinsed withdistilled water and dried in air at 55° C. for 24 hours.

For fluorescence detection, the filter membrane should have lowauto-fluorescence in the signal detection wavelength region to minimizethe background. For calorimetric and luminescence analysis, membraneswith white appearance would be preferred.

In a preferred embodiment, the filter membrane is a 0.3-0.7 mm thick,glassfiber disc filter with pore size range from 0.3 to 2.7 μm. In oneembodiment, the filter membrane is a 0.3-0.5 mm thick glassfiber discfilter with 0.7 μm pore size. In one embodiment, the filter membrane isa 0.3-0.5 mm thick glassfiber disc filter with 1.6 μm pore size. Inanother embodiment, the filter membrane is a 0.3-0.7 mm thick glassfiberdisc filter with 2.7 μm micrometer pore size.

In another preferred embodiment, the filter membrane is a nitrocelluloseor mixed cellulose ester (MCE) membrane with pore size range from 0.5 to5.0 μm. In one embodiment, the filter membrane is a 0.1-0.2 mm thick MCEmembrane with 1.6 μm pore size. In one embodiment, the filter membraneis a 0.1-0.2 mm thick MCE membrane with 3 μm pore size. In anotherembodiment, the filter membrane is a 0.1-0.2 mm thick MCE membrane with5 μm pore size.

In another preferred embodiment, the filter membrane is a nylon membranewith pore size range from 0.5 to 5.0 μm. In one embodiment, the filtermembrane is a 0.05-0.2 mm thick nylon membrane with 5 μm pore size. Inone embodiment, the filter membrane is a 0.05-0.2 mm thick MCE membranewith 10 μm pore size. In another embodiment, the filter membrane is a0.05-0.2 mm thick MCE membrane with 20 μm pore size.

The filter membrane can be bigger than the well bottom opening 11, andin close contact with the edge of the bottom well opening to effectivelyblock the microspheres or liquid from leaking through. This can beachieved by general bonding method or by support from the componentsplaced underneath the membrane. In one instances, the support is fromthe absorbing plug 20 through the friction force generated between theabsorbing plug and the housing 14.

The third component is an absorbing plug 20 in contact with the filtermembrane. Any hydrophilic absorbent material can be used as an absorbingplug, such as glassfiber, cellulose acetate fibers, sponge, foam,cotton, porous polymers or polymer fiber, such as polyethylene (PE) andpolyester (PET) etc. There is no restriction on the pore size of theabsorbing plug 20, and it can be larger than the pore size of the filtermembrane 10. It would be preferred that the absorbing plug has a largepore size so that it will have a large maximum absorbing capacity,allowing a large volume of solution added into the device. Preferablythe absorbing plug is low-fouling so that free biomolecules will haveminimum adsorption on the pores while going through the absorbing plug.The low protein-binding characteristic of the absorbing material iscritical for low background detection. Preferred materials includeglassfiber, porous PE, and porous PE/PET sheath/core bonded fiber. Inpreferred embodiments, the selected material has between 30% to 90% porevolume. In one embodiment, the absorbing plug is made of porous PE/PETsheath/core bonded fiber with 60-80% porosity and fiber directionperpendicular to the face in contact with the filter membrane. Inanother embodiment, the absorbing plug is made of hydrophilic porous PEwith 60% porosity. Depending on the application and size of the device,the total liquid absorbing capacity of the absorbing plug is from about10 μl to 50 ml.

The absorbing plug is assembled in close contact and in fluidcommunication with the filter membrane. Once the filter membrane issaturated by the aqueous solution added into the device, the aqueoussolution will pass through the filter membrane and enter the absorbingplug by capillary force. Both the contacting position and the shape ofthe absorbing plug can be varied. The contact position can be under thefilter membrane as shown in FIG. 1 and FIGS. 2A-2C, or above themembrane outside the sample well, as shown in FIGS. 2D and 2E. If thecontact is under the filter membrane, a void can be created in the plugunder the lower well opening 11 and detection zone 12, as shown in FIGS.2A-2C. The cross-section of the void has a dimension equal or largerthan the lower well opening 11 and detection zone 12, and a height rangefrom about 1 mm to the height of the absorbing plug under the membrane.The embodiment, where part of the absorbing plug has no direct contactwith the filter membrane right under the lower well opening 11 anddetection zone 12, has the advantage of reducing the signal backgroundin the detection zone. The designs in FIG. 1 and FIG. 2 are onlyexamples, not to limit the scope of the invention.

Different shapes of the absorbing plug can be used. In some embodiments,the absorbing plug has a shape selected from cylindrical, cubical,rectangular prism or polygonal prism. Size of the absorbing plug can beselected to accommodate the total volume of liquid needed to beprocessed in the assays. In one preferred embodiment, the absorbing plughas a cylindrical shape (7 mm diameter×14 mm height). In anotherpreferred embodiment, the absorbing plug has an 8 mm×8 mm×8 mm cubicshape. In another embodiment, the absorbing plug is a 16 mm width×16 mmlength×10 mm height block.

Since both the filter membrane and the absorbing plug are porousmaterial, they can be made of one or more layers of the same ordifferent materials or even merge into one piece. For example, a devicecan have a porous PE frit as the filter membrane and a porous PE rod asthe absorbing plug. In another example, a device can have multiplelayers of glassfiber discs, with the top one or more layer(s) serving asthe filter membrane and the lower ones as the absorbing plug. In anotherexample, a device can have one single rod of PE working as both thefilter membrane and the absorbing plug. In some embodiments, theabsorbing plug can be made with multiple layers of porous material, witheach layer having the same or different material, pore size, property,thickness, and/or shape. In some embodiments, the 1-piece absorbing plugcan have different material properties, such as pore size, in differentarea.

The three components of the device, sample well, filter membrane, andabsorbing plug, are hold together in close contact by friction force orby any bonding method, including, but not limiting to welding oradhesion, inside an enclosing housing 14 (FIG. 1). The housing can bemade of a single piece of plastic material such as polypropylene (PP) orpoly(methyl methacrylate) (PMMA) by injection molding, or multiple partsassembled together by adhesion, welding, or any other bonding method. Ina preferred embodiment, the housing and the sample well is made in onepiece of PP or PMMA by injection molding. And the filter membrane andabsorbing plug is fixed inside the housing under the sample well byfriction force. Other than the upper well opening 11, the case shouldhave at least one other air permeable opening that can provideventilation for the absorbing plug 20. In a preferred embodiment, asshown in FIG. 1, the bottom of the absorbing plug is sealed inside thehousing 14 with an air permeable hydrophobic membrane 15, such as thehydrophobic acrylic copolymer membrane from Pall Corporation. In anotherpreferred embodiment, the bottom of the absorbing plug is open to air.

In order to be compatible with commonly-used plate reader for signaldetection and analysis, multiple devices are arranged in a plate 60 in arectangle array format such as 8×12 (FIGS. 3A-3D), 6×8 (FIG. 3E), 4×6(FIG. 3F), or other format that user find convenient. Individual devices78 (FIG. 3B) or multiple devices pre-assembled in a smaller array blocksuch as an array of 8×1 (strip 100 in FIG. 3C) or 1×12 (strip 120 inFIG. 3D) devices, can be inserted into the plate by the user. The platecontaining one or more devices can be inserted into a plate reader or animaging instrument 170 for signal detection (FIG. 4), and be processedin a robotic high throughput work station. In a preferred embodiment,the footprint of the plate conforms to the ANSI/SLAS 2-2004 standard 96well format, with 127.76 mm length, 85.48 mm width, 14.35 mm height, and9.0 mm inter-well distance. In another preferred embodiment, thefootprint of the plate has 127.76 mm length, 85.48 mm width, and 12.0and 18.0 mm inter-well distance for 48 well (6×8 array) and 24 well (4×6array) plate respectively. In certain embodiments, as used herein, theinter-well distance means the distance between the centers of twoadjacent wells or devices.

An embodiment of the device described in this invention is shown in FIG.5. One to twelve 8-well strips each containing 8 units of the devicescan be inserted into a strip holder frame, which conforms to theANSI/SLAS 2-2004 standard 96 well format [127.76 mm length×85.48 mmwidth×14.35 mm height]. Inter-well distance is 9.0 mm. The 8-well stripsare made of opaque PMMA by injection molding. Each sample well has around upper opening with diameter 7.0 mm. The well is 3.5 mm in heightand has a curved wall. The bottom of the well has a detection zone 2 mmin diameter and 0.65 mm in height. The filter membrane under thedetection zone is a borosilicate glassfiber membrane with pore size of0.7 μm and thickness of 0.5 mm. The absorbing plug under the glassfibermembrane is a porous PE/PET fiber cube 8.0 mm(width)×7.6 mm(length)×8.0mm (height) with 70% porosity.

In some embodiments, the present invention provides a method forassaying a biological sample. The method includes filtering a mixturethrough an apparatus or a device and detecting a reporter agent with ananalytical means. In some embodiments, the mixture includes a pluralityof functionalized beads, a sample comprising a target analyte, and adetectable reporter agent. In some embodiments, the mixture includes aplurality of functionalized beads, and a sample comprising an analyte,which by itself is a reporter agent capable of producing detectablesignals. The device includes a filtering member having an upper surfaceand a lower surface; a sample well comprising a top opening for sampleaddition and a bottom opening, where the bottom opening of the samplewell is disposed on the upper surface of the filtering member anddefines a detection zone; a porous absorbing member having a surface,where the surface of the absorbing member is in contact with thefiltering member; and a housing, where the filtering member, the samplewell and the absorbing member are disposed in the housing.

In some embodiments, provided is a method for assaying a sample. Themethod includes providing a device as described herein; optionallyadding a blocking buffer to the filter membrane of the device; filteringa mixture of beads and other reagents through the filter membrane fromthe sample well in the device; adding rinsing buffer into the samplewell in the device and let the liquid be absorbed by the absorbing plugin the device; optionally adding additional samples, reaction agents,and buffers into the sample well in the device and let the liquid beabsorbed by the absorbing plug in the device; and detecting a signal,such as an optical or radioactive signal from the detection zone in thedevice.

In some embodiments, the mixture contains a plurality of functionalizedbeads, a sample containing a target analyte, and a detectable reporteragent, such as a labeled antibody or nucleic acid. In some embodiments,the mixture contains a plurality of functionalized beads, and a samplecontaining an analyte, which by itself is a reporter agent capable ofproducing detectable signals. In some embodiments, the reaction reagentsinclude unlabeled biomolecules or biomolecules conjugated with an enzymesuch as horseradish peroxidase (HRP) or alkaline phosphatase (AP), or adetectable agent such as fluorophores and micro- or nano-sizedparticles. In some embodiments, the reaction reagents include substratesfor enzyme reaction to generate a detectable signal, such aschemiluminescent, chemifluorescent, and chromogenic signal. In someembodiments, the step of detecting includes visual inspection and theuse of an analytical means including a plate reader or an imagingsystem. Exemplary plate reader includes a fluorescence plate reader or achemiluminescence plate reader. Exemplary imaging system includes acamera or a gel imager.

In certain embodiments, provided is a method for assaying a sample. Themethod includes providing a device as described herein; immobilizing alabeled or unlabeled biomolecule on a section of the filter membrane ofa device as described herein; adding a blocking buffer into the samplewell and let the liquid be absorbed through the filter membrane by theabsorbing plug of the device; adding one or more sample solution,reaction reagents and buffers into the sample well of the device and letthe liquid be absorbed through the filter membrane by the absorbing plugof the device; and detecting a signal, such as an optical or radioactivesignal from the detection zone of the device.

In some embodiments, the immobilizing step includes adding a solutioncontaining a labeled or unlabeled biomolecule such as protein or nucleicacid onto the filter membrane and drying the filter membrane. In someembodiments, the immobilizing step includes adding a solution containinga labeled or unlabeled biomolecule such as protein or nucleic acid ontothe filter membrane and let it incubate for a certain amount of time.

In some embodiments, the reaction reagents include unlabeledbiomolecules or biomolecules conjugated with an enzyme such ashorseradish peroxidase (HRP) or alkaline phosphatase (AP), or adetectable agent such as fluorophores and micro- or nano-sizedparticles. In some embodiments, the reaction reagents include substratesfor enzyme reaction to generate a detectable signal. In someembodiments, the step of detecting includes visual inspection and theuse of an analytical means including a plate reader or an imagingsystem. Exemplary plate reader includes a fluorescence plate reader or achemiluminescence plate reader. Exemplary imaging system includes acamera or a gel imager.

In an example bead-based assay, the functionalized beads 40 are firstmixed and incubated with appropriate biological sample and reagents tohave biochemical reaction occur on the surface of the beads. As a resultof the reaction, in the bead and reagent mixture, molecules that cangenerate a detectable signal, such as fluorescent, colorimetric orluminescent signal, are immobilized on the bead surface. To separate thebeads from the solution, the mixture is added into well 30. To minimizednon-specific binding of the unbound signal-generating molecules, ablocking solution such as 5% Bovine Serum Albumin can be applied to themembrane 10 prior to sample addition. As the liquid being absorbed bythe filter membrane 10 and the absorbing plug 20 through the wellopening 11, the beads aggregate in the detection zone 12. Additionalbuffer is then added into the well to further rinse the beads. The beadsare now ready for signal detection. In some applications, enzymesubstrates can be added into the detection zone to generate a detectablesignal. Depending on the type of signal, the beads can be analyzed indifferent analyzers. For example, fluorescent signal can be detected ina fluorescent plate reader. Chemiluminescent signal can be detected in aluminometer plate reader with a photo-multiplier tube (PMT) or a gelimager with a CCD camera. Colorimetric signal can be directly visualizedby naked eye or by measuring the color intensity using epi-white lightillumination. The beads in the detection zone can also be imaged on amicroscope. After use, the devices are not re-usable and can be disposedproperly.

In an example membrane-based dot blot assay, a small amount (1-5 μl) ofantigen to be probed is first added onto the filter membrane 10 and letincubate for 20 minutes. A blocking solution such as 10% Bovine SerumAlbumin is then applied to the membrane 10 and incubates for another 20minutes. A small amount (1-5 μl) of primary antibody is added onto thefilter membrane 10. After a 5-minute incubation, unbound primaryantibody is rinsed by the rinsing buffer added in to the sample well andabsorbed by the absorbing plug. A small amount (1-5 μl) of secondaryantibody conjugated with a detectable agent such as HRP, AP,fluorophore, or nano- or micro-sized particles is added onto the filtermembrane. After another 5-minute incubation, unbound secondary antibodyis rinsed by 200 μl rinsing buffer added in to the sample well andabsorbed by the absorbing plug. In some applications, enzyme substratesare added into the detection zone to generate a detectable signal.Depending on the type of signal, the membranes can be analyzed indifferent analyzers. For example, fluorescent signal can be detected ina fluorescent plate reader. Chemiluminescent signal can be detected in aluminometer plate reader with a photo-multiplier tube (PMT) or a gelimager with a CCD camera. Cotorimetric signal can be directly visualizedby naked eye or by measuring the color intensity using epi-white lightillumination. After use, the devices are not re-usable and can bedisposed properly.

The following examples are for illustrative purposes only and theinvention is not limited to the disclosed.

Example 1: Human Alpha-Fetoprotein (AFP) Fluorescence Immunoassay

This example shows how a device described in this invention can be usedfor a rapid silica bead-based fluorescent immunoassay to detect AFP fromhuman serum sample. The process can be better understood with referenceto FIG. 6.

The assay will need the following reagents: (1) silica beads 200functionalized with AFP capture antibody 210; (2) fluorescently-labeledAFP detection antibody 220; (3) serum sample containing the target AFPmolecule 230; and (4) rinsing buffer.

The capture antibody, mouse monoclonal anti-human AFP antibody, isavailable from R&D Systems. It can be covalently attached to 5 μmcarboxylated silica beads through carboxyl-amine crosslinking reaction.Functionalized silica beads can be blocked with 10% bovine serum albumin(BSA) and stored at 1% solid in phosphate buffered saline (PBS) solutioncontaining 1% BSA with 0.5% Teen-20 (1% BSA-PBST). The detectionantibody, polyclonal chicken anti-AFP antibody can be labeled withfluorophore Alexa Fluro 647 (AF647) using commercially availablelabeling kits from. Thermo Scientific. The rinsing buffer is 1%BSA-PBST.

For this assay, the device will have a sample well which is 6.5 mm indiameter, 3.5 mm in height, a detection zone which is 2 mm in diameterand 1 mm in height, 7 mm diameter glassfiber filter membrane disc with2.7 μm pore size, and an 8 mm×8 mm×8 mm porous polyethylene absorbingcube with 60-70% pore volume. Devices assembled in 8-well strip formatwith inter-device distance of 9 mm are inserted into a 96 well plateframe. Before the assay, the glassfiber filter membrane is blocked withSuperBlock blocking buffer.

A mixture of 10 μl serum sample to be examined, 10 μl anti-AFP silicabeads, and 1 μl 100 nM AF647-antiAFP antibody will be incubated at roomtemperature for 30 minutes. Multiple assays with different samples andpositive and negative controls can be performed simultaneously, 20 μl ofeach assay mixture can be added into the well of a device described inthis invention. After the solution is absorbed, silica beads will settlein the detection zone. To rinse, 200 μl rinsing buffer can be added intothe well drop by drop using a pipette. After all the PBS buffer isabsorbed, the plate can be inserted into a fluorescent plate reader forsignal detection. The plate reader should be configured into atop-reading mode, with appropriate setting of excitation and emissionwavelengths for AF647 detection. The reading should be obtained within10 minutes after the rinsing buffer is fully absorbed. The concentrationof AFP in the sample can be obtained by referring to a calibration curveobtained under the same assay condition using AFT standards.

Example 2: Human Alpha-Fetoprotein (AFP) Chemiluminescence Immunoassay

This example shows how a device described in this invention can be usedfor a rapid agarose bead-based chemiluminescent immunoassay. The processcan be better understood with reference to FIG. 7.

The assay will need the following reagents: (1) Agarose beads 300functionalized with AFP capture antibody 310; (2) AFP detection antibodylabeled with horse radish peroxidase (HRP-antiAFP antibody) 320; (3)serum sample that may contain the target AFP molecule 330; (4) rinsingbuffer 1% BSA-PBST; and (5) HRP chemiluminescence substrate 340.

The capture antibody, such as the mouse monoclonal anti-human AFPantibody from R&D Systems can be crosslinked to 20 μm glyoxal agarosebeads through glyoxal-amine chemistry. The anti-AFP agarose beads can bestored at 20% slurry 1% BSA-PEST. The detection antibody, polyclonalchicken anti-AFP antibody can be labeled with HRP using commerciallyavailable labeling kits. In some cases, HRP conjugated antibodies can bepurchased directly.

The device used for this assay will be the same as the one used inExample 1.

A mixture of 10 μl serum sample to be examined, 10 μl anti-AFP agarosebeads, and 2.5 μl 20 nM AF647-antiAFP antibody 230 will be incubated atroom temperature for 30 minutes. Multiple assays with different samplesand positive and negative controls can be performed simultaneously. 10μl of each assay mixture can be added into the well of a devicedescribed in this invention. After the solution is absorbed, agarosebeads will settle in the detection zone with a slurry volume of 2 μl. Torinse away the free HPR-antibodies, 50 μl rinsing buffer can be addedinto the well using a pipette for 4 times, 200 μl total. After all therinsing buffer is absorbed, 10 μl HRP chemiluminescent substrate can beadded onto the agarose beads. Different commercially availablechemiluminescent substrate can be used, such as the Clarity Western ECLsubstrate from Bio-Rad, or SuperSignal West Femto ChemiluminescentSubstrate from Thermo Scientific. Immediately after 10 minute incubationat room temperature, the plate can be inserted into a luminometer platereader for signal detection. The plate reader should be configured intoa top-reading mode, with appropriate setting for luminescence detection.The chemiluminescent signal can also be detected and imaged in a gelimaging system. The concentration of AFP in the sample can be obtainedby referring to a calibration curve obtained under the same assaycondition using AFP standards.

Example 3: Botulinum Neurotoxin Type A (BoNT/A) Fluorescence ResonanceEnergy Transfer (FRET) Assay

This example shows how the device in this invention can be used torapidly detect active BoNT/A in a large volume of serum sample usingpolyacrylamide beads and a FRET substrate (see FIG. 8).

The assay will need to use the following reagents: (1) Polyacrylamidebeads 400 functionalized with anti-BoNT/A antibody 410; (2) serum samplethat may contain the active BoNT/A molecule 420; (3) assay buffer 20 mMHEPES at pH 8.0; and (4) SNAPtide fluorogenic BoNT/A substrate 430 fromEMD Millipore.

BoNT/A antibody (Santa Cruz Biotechnology) can be crosslinked topre-activated polyacrylamide (PA) beads such as Ultralink Biosupportfrom Thermo Scientific. The anti-BoNT/A PA beads can be stored at 20%slurry in PBS solution containing 1% BSA with 0.5% Tween-20. 10 μlanti-BoNT/A PA beads will be added into 500 μl serum sample andincubated at 37° C. for 1 hour with slow rotation. The mixture will beadded into a device described in this invention. For this assay, thedevice will have well which is 7 mm in diameter, 5 mm in height, adetection zone which is 2 mm in diameter and 1 mm in height, 7 mmdiameter and 0.5 mm thick glassfiber membrane disc with 2.7 micrometerpore size, and an 8 mm×8 mm×20 mm porous polyethylene absorbing plugwith 60-70% pore volume. Devices assembled in 8-well strip format withinter-device distance of 9 mm are inserted into a 96 well strip frame.

Multiple assays can be performed simultaneously. After the 500 μl samplesolution is totally absorbed, the PA beads with captured BoNT/A targetcollected in the detection zone can be rinsed with 200 μl assay buffer.Finally, 10 μl SNAPtide substrate at appropriate working concentration,typically between 5-10×10⁻⁶ M, will be added onto the beads. The platecan be placed inside a 37° C. humidified incubator for 10 minute for thereaction, after which the FRET signal will be measured from the plate.If active BoNT/A is present on the beads, the FRET substrate will becleaved and the fluorophore will be released. Fluorescent intensitymeasured with excitation wavelength at 320 nm and emission wavelength at420 nm will indicate the concentration of active BoNT/A in the samplewith reference to a standard curve.

Example 4: Human Interleukin-6 (IL6) Fluorescence Immunoassay

This example shows how a device described in this invention was used toobtain a standard calibration curve for a rapid fluorescent immunoassayto detect target protein from human serum sample.

The device used for this assay will be the same as the one used inExample 1. The reagents used in this assay include the following: (1)Agarose beads functionalized with anti-human IL6 antibody (Agr-antiIL6);(2) Alexa Fluor 647-labeled anti-human IL6 antibody (AF647-antiIL6); (3)recombinant human IL6 as assay standard; and (4) 1% BSA-PBST as dilutionbuffer and rinsing buffer.

Agr-anti IL6 was prepared using glyoxal agarose beads (Agarose BeadsTechnologies) and goat anti-IL6 polyclonal antibody (R&D Systems)according to manufacturer's instructions. They were stored in PBST-1%BSA buffer at 20% bead volume before use. AF647-anti IL6 was preparedwith Alexa Fluor® 647 Antibody Labeling Kit (Life Technologies) and goatanti-IL6 polyclonal antibody (R&D Systems) according to manufacturer'sinstructions.

A 10-fold serial dilution of the human IL6 was prepared using thePBST-1% BSA dilution buffer. For each concentration of the IL6 standard,10 μl IL6, 10 μl Agr-anti IL6, and 1 μl 100 nM AF647-anti IL6 were mixedtogether in a 1.5 ml centrifuge tube. Multiple reactions were startedsimultaneously. After 30 min incubation at room temperature, 20 μl ofeach reaction mix was added into the sample wells of the device. Afterthe solution was absorbed, agarose beads settled in the detection zone.To rinse, 50 μl rinsing buffer was added into the well and this wasrepeated for 4 times. A total of 200 μl rinsing buffer was used andfully absorbed by the device. The plate was then inserted into afluorescent microplate reader (TECAN) for signal detection. The platewas configured into a top-reading mode, with excitation wavelength at652 nm and emission wavelength at 680 nm, and light beam focused at thedetection zone.

The assay results with IL6 concentration ranging from 10 pg/ml to 100ng/ml are displayed in FIG. 9. The fluorescent signal detected from theagarose beads increased as the concentration of IL6 increases. Thestandard curve can be used to find out the IL6 concentration in anunknown sample. The entire assay only took 30 min of incubation time andless than 10 min hands-on processing time, without using vacuum, magnet,centrifugation, etc., just pipetting solution into the device. TheFluorescent signal level was stable for at least 10 min.

Example 5: Mouse Immunoglobulin G (NC) Chemiluminescent Dot Blot

This example shows how a device described in this invention was used fora rapid chemiluminescent dot blot assay to probe a mouse IgG dissolvedin PBS.

This example use the same embodiment of the device as described inExample 1, except that the filter membrane is an untreated glassfiberfilter membrane with 0.7 μm pore size.

The assay process is illustrated in FIGS. 10A and 10B. Mouse IgG (R&DSystems) 500 was diluted in PBS to make 100 ng/ml, 10 ng/ml and 1 ng/mlsamples. 1 μl of each sample was directly spotted onto the center of theuntreated glassfiber membrane inside the well. The membrane was let dryin air for 20 min. 50 μl of 10% BSA 510 was added into each well and letsit for 15 mm for blocking. For probing, 1 μl of 5 nM goat anti-mouseIgG conjugated with horse radish peroxidase (goat anti-mouse IgG-HRP)(KPL) 520 was added onto the membrane and let incubate for 5 min. Torinse, 50 μl of PBST-1% BSA 530 was added into the well and let absorbedcompletely. This step was repeated for 4 times. Immediately after 10 μlof chemiluminescentHRP substrate 540, SuperSignal Fe/MOD (ThermoScientific), was added into each well, the device was placed inside adigital gel imaging system (ProteinSimple) for a chemiluminescent (CL)image obtained with 1 second exposure time.

The resulting CL image is shown in FIG. 11. For increasing amount ofmouse IgG spotted onto the membrane, there are stronger CL signal fromthe sample well. The sensitivity of the assay is comparable to aconventional dot blot. The entire process took less than an hour, muchless than hours or overnight procedure using conventional methods.

What is claimed is:
 1. A device for biomolecule analysis, said devicecomprising: a filter membrane having an upper surface and a lowersurface; a sample well comprising a top opening for sample addition anda bottom opening, wherein the bottom opening of said sample well isdisposed on the upper surface of said filter membrane and defines adetection zone, wherein the detection zone has a height from about 0.2mm to about 2 mm; a porous absorbing plug having a surface, wherein thesurface of the absorbing plug is in contact with the filter membrane andwherein the absorbing plug is a hydrophilic porous material having fromabout 30 to about 90 percent pore volume, with liquid absorbing capacityfrom about 10 μl to about 50 ml; and a housing, wherein the filtermembrane, the sample well and the absorbing plug are disposed in saidhousing.
 2. The device of claim 1, wherein the absorbing plug has abottom surface, which is in contact with a hydrophobic membrane.
 3. Thedevice of claim 1, wherein the cross section of the detection zone has ashape selected from the group consisting of circle, oval, triangle,square, rectangle, pentagon, hexagon and octagon with sharp or roundedcorner, or a combination thereof.
 4. The device of claim 1, wherein thesample well has a sloped inner surface around the bottom opening, andwherein the volume of the sample well is from about 20 μl to about 10ml.
 5. The device of claim 1, wherein the cross-section of the samplewell has a shape selected from the group consisting of circle, oval,square, rectangle, pentagon, hexagon and octagon, with sharp or roundedcorner, or a combination thereof.
 6. The device of claim 1, wherein thesample well is made of a material selecting from the group consisting ofpolypropylene, poly(methyl methacrylate) (PMMA), polystyrene,acrylonitrile butadiene styrene (ABS), polycarbonate (PC), nylon orcombinations thereof.
 7. The device of claim 1, wherein the filtermembrane is a hydrophilic porous membrane having a plurality of voidspaces or pores, wherein the plurality of void spaces has a dimensionranging from about 0.2 μm to about 50 μm.
 8. The device of claim 1,wherein the filter membrane is selected from the group consisting of asilver membrane, a glassfiber filter membrane, a nitrocellulosemembrane, a mixed cellulose ester (MCE) membrane, a polycarbonate (PC)membrane, a polyester (PE) membrane, a cellulose acetate membrane, anylon membrane, a polyethersulfone (PES) membrane, a polyvinylidenedifluoride (PVDF) membrane, a regenerated cellulose (RC) membrane,porous polyethylene (PE) membrane and porous polypropylene (PP)membrane.
 9. The device of claim 1, wherein the filter membrane is aborosilicate glassfiber filter membrane having a plurality of pores,wherein the plurality of pores has a pore size ranging from about 0.3 μmto about 5.0 μm.
 10. The device of claim 1, wherein the filter membraneis a nitrocellulose or mixed cellulose ester (MCE) filter membranehaving a plurality of pores, wherein the plurality of pores has a poresize ranging from about 0.2 μm to about 5 μm.
 11. The device of claim 1,wherein the filter membrane is a nylon membrane having a plurality ofpores, wherein the plurality of pores has a pore size ranging from about0.45 μm to about 5 μm.
 12. The device of claim 1, wherein the filtermembrane is a porous membrane treated with one or more blocking agents,wherein the blocking agents bind to the surface of the membrane throughadsorption or chemical cross-linking.
 13. The device of claim 1, whereinthe absorbing plug is in contact with the lower surface of the filtermembrane.
 14. The device of claim 13, wherein the absorbing plugcomprises one or more voids situated beneath the bottom opening of thesample well, with a cross-section dimension equal or greater than thatof the bottom opening of the sample well, and a height from about 0.5 mmto the height of the absorbing plug under the membrane.
 15. The deviceof claim 1, wherein the absorbing plug is in contact with the uppersurface of the filter membrane and wherein the portion of the filtermembrane in contact with the absorbing plug is located outside thesample well.
 16. The device of claim 1, wherein the cross section of theabsorbing plug has a shape selected from the group consisting of circle,oval, square, rectangle, pentagon, hexagon and octagon, each of whichhas a sharp or rounded corner or a combination thereof.
 17. The deviceof claim 1, wherein the absorbing plug is made of one or morehydrophilic porous materials selected from the group consisting ofglassfiber, cellulose acetate fibers, cotton, porous polyethylene (PE),and polyethylene (PE)/polyester (PET) sheath/core fiber.
 18. The deviceof claim 1, wherein a portion of the absorbing plug is made ofhydrophilic polyethylene (PE)/polyester (PET) sheath/core bonded fiber,with the fiber aligned in a direction perpendicular to the surface ofthe filter membrane.
 19. The device of claim 1, wherein the housing,sample well, filter membrane and absorbing plug are placed together incontact through injection molding, friction, or a bonding methodselected from welding or adhesion.
 20. The device of claim 1, whereinthe housing and the sample well are molded into one piece.
 21. Thedevice of claim 1, wherein the filter membrane and absorbing plug areblended into a single component.
 22. The device of claim 1, wherein thecross section of the detection zone has a dimension from about 0.2 mm²to about 38 mm².
 23. A plate for biomolecule analysis comprising arectangular array of fixed or removable devices, each of the devicescomprising: a filter membrane having an upper surface and a lowersurface; a sample well comprising a top opening for adding sample and abottom opening, wherein the bottom opening of said sample well isdisposed on the upper surface of said filter membrane and defines adetection zone, wherein the detection zone has a height from about 0.2mm to about 2 mm; a porous absorbing plug having a surface; wherein thesurface of the absorbing plug is in contact with the filter membrane andwherein the absorbing plug is a hydrophilic porous material having fromabout 30 to about 90 Percent yore volume, with liquid absorbing capacityfrom about 10 μl to about 50 ml; and a housing, wherein the filtermembrane, the sample well and the absorbing plug are disposed in saidhousing.
 24. The plate of claim 23, having a width of about 127.8 mm,length of about 85.5 mm, and height ranging from about 14 mm to about 30mm.
 25. The plate of claim 23, wherein the distance between two adjacentdevices is from about 3 mm to about 30 mm.
 26. The plate of claim 25,wherein the plate comprises devices arranged in a rectangular 1×1, 8×1,8×2, 1×12, 2×12, or 8×12 format, wherein the distance between twoadjacent devices is about 9 mm.
 27. The plate of claim 25, wherein theplate comprises devices arranged in a rectangular 1×1, 6×1, 1×8, or 6×8format, wherein the distance between the adjacent devices is about 12mm.
 28. The plate of claim 25, wherein the plate comprises devicesarranged in a rectangular 1×1, 4×1, 1×6, and 4×6 format, wherein thedistance between the adjacent devices is about 18 mm.
 29. The plate ofclaim 25, wherein the plate comprises devices arranged in a rectangular16×24 format, wherein the distance between the adjacent devices is about4.5 mm.
 30. The plate of claim 23, wherein the plate comprises the arrayof devices arranged in a rectangular format selected from the groupconsisting of 1×1, 8×1, 8×2, 1×12, 2×12, 8×12, 6×1, 1×8, 6×8, 4×11, 1×6,4×6, and 16×24 format.
 31. The plate of claim 23, wherein the crosssection of the detection zone has a dimension from about 0.2 mm² toabout 38 mm².